Without limiting the scope of the invention, its background is described in connection with fluorescence spectroscopy and microscopy fluorescence. Fluorescence is a phenomenon that has provided many useful applications and fluorescence spectroscopy is a rapidly developing and crucial component in the areas of flow cytometry, medical diagnostics, DNA sequencing, genetics and cellular and molecular imaging. For example, fluorescent beads have provided a new way in which to produce new assays, e.g., polystyrene beads that enclosed temperature-sensitive fluorophores and fluorescent bead manufactured by coating fluorophores on the outside surface of polystyrene particles. However, the random distribution of dye and high dye to nanoparticle ratio, the fluorescence polarization observed from the beads is low.
Most fluorescent molecules or objects (like quantum dots and fluorescent beads) are indispensible for testing and calibrating instrumentation. In microscopy the typical dyes have problems like photobleaching and/or blinking. Because of that more stable emitter systems like quantum dots or microspheres (beads) are widely used, very stable optically polymer-core nanoparticles with immobilized dyes also proved their utility in studding live biological systems with dynamic flow tracking because of their spectral properties with usually high quantum yield, extinction coefficient and photostability. Nanospheres are widely employed now in the tissue imaging, biotechnology, as temperature sensors and have been used in a variety of applications that include diagnostics and biological assays. However, a high local density of fluorescent molecules enforces a depolarization of observed fluorescence. As a result, these fluorescent nanoparticles cannot be used for studies that utilize polarization methods.